Project Summary Heart diseases including ischemic myocardial infarction (MI) continue to be the leading cause of mortality in the U.S. While reperfusion therapy such as percutaneous coronary intervention remains the most effective strategy to limit infarct size and preserve cardiac function, reperfusion itself can cause lethal injury to myocardium. In response to ischemia-reperfusion (I/R), myocardium releases various danger molecules that are proven to be harmful to the heart. We have recently identified that cellular RNAs including many microRNAs (miRNAs) are released from the heart during I/R and that extracellular RNA contributes to myocardial inflammation and injury. Our preliminary investigations demonstrate that RNA isolated from the heart or single-stranded miRNA mimics (miR-34, -122, -133a, -142, 146a, -208a) induce a robust cytokine response in cardiomyocytes, neutrophils, and macrophages. Using genetic deletion models and pharmacological inhibitors, we demonstrate, in cell-based assays, that cardiac RNAs or microRNA mimics induce cytokine production specifically through the innate immune Toll-like receptor 7 (TLR7) signaling. The over goals of this proposal are to determine the role of extracellular miRNAs in myocardial I/R injury and explore the underlying molecular mechanisms. The proposal is based on the following three hypotheses: 1) that miRNAs are released from injured myocardium during I/R and play a role in myocardial inflammation and injury, 2) that miRNAs act through TLR7 signaling in cardiomyocytes and neutrophils, 3) that pharmacological inhibition of miRNAs and TLR7 signaling after ischemic insult will offer cardio-protection against I/R injury. In Specific Aim 1, we will determine the contribution of extracellular miRNAs to myocardial inflammation and injury during I/R. In Specific Aim 2, we will test the role of TLR7 signaling in miRNA-mediated myocardial inflammation and injury in I/R. In Specific Aim 3, we will explore the miRNA-TLR7 signaling as a potential therapeutic target for the treatment of myocardial I/R injury. The proposed studies address a unique and novel function of extracellular miRNAs in myocardial inflammation and injury, with significant implications in pathogenesis and treatment of ischemic myocardial infarction.